Prevention and alleviation of steric hindrance during single molecule synthesis

ABSTRACT

The present invention provides compositions and methods for reducing steric hindrance in the product of nucleic acid polymerase reaction. Methods and compositions of the invention encompass application of exonucleases, endonucleases, and uracil-DNA glycosylases to a nucleic acid polymerase reaction such that newly formed nucleic acid strands are modified (e.g., cleaved) while the polymerase reaction continues to proceed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/535,385, filed Aug. 4, 2009, which claims the benefit of U.S.Provisional Application No. 61/086,404, filed Aug. 5, 2008, thedisclosures of both of which are incorporated herein by reference intheir entireties for all purposes.

FIELD OF THE INVENTION

The present invention is generally related to the field of molecularbiology. The present invention provides compositions and methods forreducing steric hindrance caused by the product of a nucleic acidpolymerase reaction. Methods and compositions of the invention encompassbut are not limited to application of exonucleases, endonucleases, andglycosylases to the product of a nucleic acid polymerase reaction suchthat newly formed nucleic acid strands or the portions of the templateto which they are annealed are modified while the polymerase reactioncontinues to proceed along the template nucleic acid.

BACKGROUND OF THE INVENTION

During nucleic acid polymerase reactions, as the polymerase enzymeproceeds along the template, a nascent nucleic acid strand is generated.When such polymerase reactions are conducted in restrictive volumes(i.e., less than 1 μl), steric hindrance problems can arise as thenascent strand grows in length. In restricted volume reactions, thenascent strand is more likely to hinder the progress of the polymeraseenzyme than in reactions taking place in less restrictive volumes.Methods for mitigating such steric hindrance would be beneficial incertain polymerase reactions, e.g., those carried out in restrictivevolumes.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for preventingand/or alleviating steric hindrance that may be caused by the product ofa nucleic acid polymerase reaction. For example, one strategy forreducing the steric hindrance posed by the nascent strand is to removeor cleave the nascent strand while keeping the template nucleic acidintact.

In certain aspects, the present invention provides methods for reducingsteric hindrance resulting from a product of a nucleic acid polymerasereaction. For example, one such method includes the step of conductingthe nucleic acid polymerase reaction in the presence of an exonuclease.In certain embodiments of the invention, the nucleic acid polymerasereaction includes a template nucleic acid molecule, and the exonucleasecleaves the product of the reaction, e.g., the nascent nucleic acidstrand, but does not cleave the template nucleic acid molecule. Incertain related embodiments, the exonuclease is only able to initiate atthe 5′ end of a single-stranded nucleic acid, e.g., the product of a“rolling-circle” replication reaction in which a circular nucleic acidserves as the template. Certain preferred exonucleases for use with theinstant invention include RecJ, lambda exonuclease, and T7 exonuclease.In certain preferred embodiments, the polymerase reaction is conductedin a volume of less than one attoliter.

In another embodiment of the present invention, a method for reducingsteric hindrance resulting from a product of a nucleic acid polymerasereaction includes the step of conducting the nucleic acid polymerasereaction in the presence of a restriction endonuclease. In thisembodiment, the nucleic acid polymerase reaction includes a templatenucleic acid molecule, and the restriction endonuclease cleaves theproduct of the nucleic acid polymerase reaction but does not cleave thetemplate nucleic acid molecule. In certain preferred embodiments, therestriction enzyme has a four-base-pair recognition site. In furtherpreferred embodiments, the polymerase reaction is conducted in a volumeof less than one attoliter.

In another embodiment of the present invention, a method for reducingsteric hindrance in the product of a nucleic acid polymerase reactionincludes the step of conducting the nucleic acid polymerase reaction inthe presence of a uracil-DNA glycosylase enzyme. In this embodiment, thenucleic acid polymerase reaction includes a template nucleic acidmolecule, and the uracil-DNA glycosylase removes uracil nitrogenousbases from the product of the nucleic acid polymerase reaction but doesnot remove nitrogenous bases from the template nucleic acid molecule. Incertain preferred embodiments, the polymerase reaction is conducted in avolume of less than one attoliter.

In certain aspects, the invention provides methods of determining anucleotide sequence of a template nucleic acid molecule. In certainembodiments, the method includes the step of conducting a nucleic acidpolymerase reaction in the presence of a set of detectableterminal-phosphate-labeled nucleotides and an exonuclease. In thismethod, the nucleic acid polymerase reaction results in the addition ofthe members of the set of detectable terminal-phosphate-labelednucleotides to a nascent nucleic acid strand and also results inproduction of a labeled polyphosphates that are released from themembers of the set of detectable terminal-phosphate-labeled nucleotides.This method further includes the step of sequentially detecting eitherthe released labeled polyphosphates or the terminal-phosphate-labelednucleotides that yield the labeled polyphosphates during the polymerasereaction, and subsequent identification of the same. Identification ofthe released labeled polyphosphates or the terminal-phosphate-labelednucleotides that yield the labeled polyphosphates determines thenucleotide sequence of the template nucleic acid. In certain embodimentsof the invention, the exonuclease cleaves the nascent nucleic acidstrand but does not cleave the template nucleic acid.

In another embodiment, the invention provides a method of determining anucleotide sequence of a template nucleic acid that includes the step ofconducting a nucleic acid polymerase reaction in the presence of a setof detectable terminal-phosphate-labeled nucleotides and a restrictionendonuclease. In this method, the nucleic acid polymerase reactionresults in the addition of detectable terminal-phosphate-labelednucleotides to a nascent nucleic acid strand and also results inproduction of labeled polyphosphates that are released from thedetectable terminal-phosphate-labeled nucleotides. This method furtherincludes the step of sequentially detecting during the polymerasereaction either the released labeled polyphosphates or theterminal-phosphate-labeled nucleotides that yield the labeledpolyphosphates, and subsequent identification of the same.Identification of the released labeled polyphosphates or theterminal-phosphate-labeled nucleotides that yield the labeledpolyphosphates determines the nucleotide sequence of the templatenucleic acid. In certain embodiments of the invention, the restrictionendonuclease cleaves a product of the nucleic acid polymerase reactionbut does not cleave the template nucleic acid.

In another embodiment, the invention provides a method of determining anucleotide sequence of a template nucleic acid that includes the step ofconducting a nucleic acid polymerase reaction in the presence of a setof detectable terminal-phosphate-labeled nucleotides, dUTP (or an analogor derivative thereof), and a uracil-DNA glycosylase enzyme. In certainpreferred embodiments, the dUTP (or analog or derivative thereof) is adUTP analog comprising at least four, five, six, seven, or eightphosphate groups. In this method, the nucleic acid polymerase reactionresults in the addition of the detectable terminal-phosphate-labelednucleotides to a nascent nucleic acid strand and also results inproduction of labeled polyphosphates that are released from thedetectable terminal-phosphate-labeled nucleotides. This method furtherincludes the step of sequentially detecting during the polymerasereaction either the released labeled polyphosphates or theterminal-phosphate-labeled nucleotides that yield the labeledpolyphosphates, and subsequent identification of the same.Identification of the released labeled polyphosphates or theterminal-phosphate-labeled nucleotides that yield the labeledpolyphosphates determines the nucleotide sequence of the templatenucleic acid. In certain embodiments of the invention, the uracil-DNAglycosylase removes uracil nitrogenous bases from a product of thenucleic acid polymerase reaction, but does not remove nitrogenous basesfrom the template nucleic acid. Further, the removal of uracilnitrogenous bases enhances flexibility of the nascent strand, andfurther increases the likelihood of hydrolytic cleavage of the phosphatebackbone of the nascent strand at the resulting abasic sites. In certainembodiments, the method further comprises hydrolysis of abasic sitescreated by removal of uracil residues from the nascent nucleic acidstrand by the uracil-DNA glycosylase. Optionally, the method can beperformed in the presence of at least one polyamine or AP endonuclease.Additionally, the set of detectable terminal-phosphate-labelednucleotides can optionally include terminal-phosphate-labeledthymine-containing nucleotides.

In certain aspects, the invention provides a template nucleic acid thatcomprises a modification such that an exonuclease, restrictionendonuclease, and/or a uracil-DNA glycosylase is unable to cleave thetemplate nucleic acid. Such modifications include but are not limited tonon-natural nucleotides at one or both termini of the template nucleicacid, methylation of the template nucleic acid, a protecting group atone or both termini of the template nucleic acid (e.g., a removableprotection group), a chemically-modified oligonucleotide extensionligated to an end of the template nucleic acid, as described elsewhereherein. For example, single-stranded template nucleic acid can beprotected by a phosphorothioate bond. In certain embodiments, thetemplate nucleic acid is immobilized within an optical confinement,e.g., a zero mode waveguide.

In certain aspects, the invention provides a composition that includes anucleic acid polymerase, a restriction endonuclease or an exonuclease ora uracil-DNA glycosylase, and a template nucleic acid. In a furtheraspect, the composition also includes a substrate. In a still furtheraspect, the nucleic acid polymerase is immobilized on the surface of thesubstrate. In a still further aspect, the substrate is part of aconstricted volume reaction vessel.

In another aspect, the invention provides a kit. Such a kit can includea substrate, which includes a surface. The kit can also include anucleic acid polymerase. In one aspect, the nucleic acid polymerase isimmobilized on the surface of the substrate. In a further aspect, thesubstrate is part of a constricted volume reaction vessel. Such a kitcan also include a restriction endonuclease, an exonucleases, oruracil-DNA glycosylases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an illustrative example of a single molecule synthesisreaction located within a restricted volume reaction vessel.

FIG. 2 illustrates certain embodiments of the invention in which anexonuclease acts upon a product of a single molecule synthesis reaction.Embodiments comprising a dsDNA-dependent ssDNA exonuclease, a dsDNAexonuclease, and a ssDNA-dependent ssDNA exonuclease are shown in A, B,and C, respectively.

FIG. 3 illustrates certain embodiments of the invention in which anendonuclease acts upon a product of a single molecule synthesisreaction. Embodiments comprising a dsDNA-dependent ssDNA endonuclease, adsDNA endonuclease, and a ssDNA-dependent ssDNA endonuclease are shownin A, B, and C, respectively.

FIG. 4 illustrates certain embodiments of the invention in which auracil-DNA glycosylase acts upon a product of a single moleculesynthesis reaction. Embodiments comprising a linear ssDNA template and acircular ssDNA template are shown in A and B, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, formulations and methodologies whichare described in the publication and which might be used in connectionwith the presently described invention.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention. It will be apparent to one of skill inthe art that these additional features are also encompassed by thepresent invention.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. The techniques and procedures are generallyperformed according to conventional methods in the art and variousgeneral references (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry and organicsynthesis described below are those well known and commonly employed inthe art. Standard techniques, or modifications thereof, are used forchemical syntheses and chemical analyses.

Overview

The present invention provides methods and compositions for preventingsteric hindrance of a nucleic acid polymerase reaction by the product ofthat same reaction. In certain embodiments, steric hindrance isprevented or alleviated by cleaving the product of a polymerasereaction. In polymerase reactions, particularly reactions taking placein small volumes, the growing nascent strand can interfere with theactivity of the polymerase enzyme as the reaction progresses. Thepresent invention encompasses methods and compositions for modifying(e.g., cleaving, fragmenting, or removing nitrogenous bases from) theproduct of a nucleic acid polymerase reaction without modifying thetemplate nucleic acid.

The term “template nucleic acid” as used herein is a nucleic acidmolecule that serves as a pattern for synthesis of a macromolecule, suchas DNA or RNA. For example, during a polymerization reaction, apolymerase enzyme can use the sequence of the template to direct thesequence of nucleotides (or analogs or derivatives thereof) incorporatedinto a nascent strand, thereby creating a nascent stand that iscomplementary to the template nucleic acid. The product of a nucleicacid reaction includes the nascent nucleic acid strand that is generatedby the polymerase enzyme. The product of a nucleic acid reaction canalso include the double-stranded molecule comprising the nascent strandannealed to the complementary region of the template nucleic acid uponwhich it was synthesized. The present invention also includes methodsand compositions for protecting the template nucleic acid from beingmodified by any enzymes or other molecules applied to reduce sterichindrance posed by the product of the nucleic acid polymerase reaction.

Nucleic Acid Polymerase Reactions

In accordance with the present invention, the compositions and methodsdescribed herein can be used to reduce steric hindrance of nucleic acidpolymerase reactions. Such steric hindrance may arise from the productof those reactions, in particular in highly processive polymerasereactions that produce long single-stranded or double-stranded nucleicacid products. The growing nascent strand can, in certain embodiments,be thousands of bases in length. For example, some polymerase reactionscomprise a circular single-stranded nucleic acid template that isrepeatedly processed by a polymerase in a “rolling-circle replication”reaction. The nascent strand produced comprises multiple copies of thecomplement of the template strand that are displaced by thetranslocating polymerase enzyme to produce a long concatemer moleculethat typically remains attached to the polymerase complex. Certain ofthe inhibitory effects of having a long nascent strand in arolling-circle replication embodiment are related to its residence on acircular template, necessitating continuous strand displacement by thepolymerase during ongoing synthesis of the nascent strand. Further,certain polymerase reactions comprise a long single-stranded nucleicacid template and form a long double-stranded product through synthesisof the complementary nascent strand on the template. Sincedouble-stranded DNA is a much more rigid structure than single-strandedDNA, the reduced flexibility of the long double-stranded productrelative to the initial single-stranded template can cause escalatingsteric hindrance of the polymerase reaction as the length of the duplexproduct increases. In addition to steric hindrances that can interferewith the ongoing polymerase reaction, a long single-stranded ordouble-stranded product attached to a polymerase complex immobilized ona surface can stress the attachment between the surface and the complex(e.g., via the polymerase enzyme or the template nucleic acid), possiblycausing displacement of the complex from the surface. In certainembodiments, such displacement effectively ends analysis of the reactionby allowing the complex to move away from the substrate and preventingfurther monitoring of the reaction by the practitioner. One of ordinaryskill will recognize additional negative impacts of having a longpolymerase reaction product present in an ongoing polymerase reaction,including but not limited to the possibility that the product may be asubstrate for a competing reaction that could confound the ability tomonitor the polymerase reaction. As such, an object of the instantinvention is to facilitate modification or removal of a polymerasereaction product during the course of the polymerase reaction, i.e.,during ongoing incorporation of nucleotides into a growing nascentstrand by a polymerase enzyme.

Steric hindrance can be particularly problematic when the nucleic acidpolymerase reactions take place in small volumes. The term “smallvolumes” as used herein refers to volumes of less than about 1 attoliter(al). In an exemplary aspect, the small volume reactions according tothe invention are in the range of about 100 zeptoliters (zl) to about 1al. In another exemplary aspect, the small volume reactions according tothe invention are about 50, 100, 150, 200, 250, 300, 350, 400, 450, and500 zl. In still another exemplary aspect, the small volume reactionsaccording to the invention are in the range of about 20 to about 1000,about 50 to about 750, about 100 to about 500, about 150 to about 450,about 200 to about 400, and about 250 to about 350 zl.

In certain embodiments, the small volumes do not refer to the volume ofan entire reaction mixture, but rather refer to a portion of thereaction mixture located within a restricted volume reaction vessel(e.g., a well, optical confinement, microfluidic channel, or otherstructure within which a reaction is occurring). For example, a reactionvolume may comprise a plurality of “small volume reactions,” each ofwhich is located within a restricted volume reaction vessel, e.g., in anarray of restricted volume reaction vessels on a substrate. Certainpreferred embodiments of arrays of restricted volume reaction vessels ona substrate and methods of use and manufacture thereof are furtherdescribed, e.g., in U.S. Pat. Nos. 7,313,308 and 7,486,865; and U.S.patent application Ser. Nos. 11/978,138, filed Oct. 26, 2007, and11/704,733, filed Feb. 9, 2007. As such, the small volume reactionsaccording to the invention are, in an exemplary embodiment, contained ina constricted volume reaction vessel, such as, in one non-limitingexample, a zero mode waveguide. Constricted volume reaction vessels,including zero mode waveguides, are known in the art and describedfurther herein. In certain preferred embodiments, a single restrictedvolume reaction vessel contains a single polymerase reaction, as furtherdescribed below.

In accordance with the invention, “small volume” nucleic acid polymerasereactions may also be defined in terms of the size of the nucleic acidstrands that are part of such reactions. Such nucleic acid strands maybe the template nucleic acid or the nascent nucleic acid. In oneexemplary embodiment, the small volume reaction may comprise a nucleicacid strand of about 500, 750, 1000, 1250, 1500, 1750, 2000, 2500, 5000,7500, 10,000, 15,000, 20,000, 50,000, 75,000, and 100,000 bases. Thesmall volume reaction may in another exemplary embodiment comprise anucleic acid strand of about 500 to about 100,000, of about 1000 toabout 50,000, of about 2000 to about 25,000, of about 3000 to about20,000, of about 4000 to about 15,000, and of about 5000 to about 10,000bases. In some embodiments, the nascent strand is longer than thetemplate nucleic acid strand, e.g., and may comprise multiple copies ofthe complement of the template nucleic acid strand. The volume of thesmall volume reaction defined by the size of the nucleic acid strandsmay be based on the relaxed sizes of these strands or on the coiled orsupercoiled sizes of these strands. As discussed further herein, suchnucleic acid strands may be single- or double-stranded, circular orlinear.

A nucleic acid polymerase is any of various enzymes, such as DNApolymerase, RNA polymerase, or reverse transcriptase, that catalyze theformation of polynucleotides of DNA or RNA using an existing strand ofDNA or RNA as a template. As such, nucleic acid polymerase reactions ofthe invention may include polymerase enzymes such as DNA polymerases,i.e., Tag polymerases, E. coli DNA polymerase 1, Klenow fragment,reverse transcriptases, Φ29 related polymerases including wild type Φ29polymerase and derivatives of such polymerases, T7 DNA polymerase, T5DNA polymerase, RNA polymerases, and the like. Further descriptions ofpolymerases are provided in U.S. patent application Ser. Nos.11/645,125, filed Dec. 21, 2006; 11/978,210, filed Oct. 26, 2007;12/384,112, filed Mar. 30, 2009; 12/384,110, filed Mar. 30, 2009; and61/094,843, filed Sep. 5, 2008; and U.S. Patent Publication Nos.20070196846 and 20080108082; the disclosures of which are incorporatedherein by reference in their entireties for all purposes.

As used herein, the term “polynucleotide” is used interchangeably with“nucleic acid” to mean DNA, RNA, single-stranded, double-stranded, ormore highly aggregated hybridization motifs, and any chemicalmodifications thereof. Modifications include, but are not limited to,those providing chemical groups that incorporate additional charge,polarizability, hydrogen bonding, electrostatic interaction, points ofattachment and functionality to the nucleic acid ligand bases or to thenucleic acid ligand as a whole. Such modifications include, but are notlimited to, peptide nucleic acids (PNAs), phosphodiester groupmodifications (e.g., phosphorothioates, methylphosphonates), 2′-positionsugar modifications, 5-position pyrimidine modifications, 8-positionpurine modifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases, isocytidine and isoguanidine and the like. Nucleic acidscan also include non-natural bases, such as, for example, nitroindole.Modifications can also include 3′ and 5′ modifications such as cappingwith a fluorophore (e.g., quantum dot) or another moiety. Templatenucleic acid molecules used in accordance with the invention may besingle-stranded or double-stranded, and may be linear or circularmolecules.

The methods of the present invention are particularly useful in reducingthe steric hindrance that may occur during “sequence-by-incorporation”reactions in which the sequence of bases in a nucleic acid is identifiedbased upon the template-dependent incorporation of complementarynucleotides by a polymerase enzyme. The term “nucleotides” as usedherein includes native ribonucleotides and deoxyribonucleotides, as wellas analogs, derivatives, mimetics, and any chemical modificationsthereof, as further described above. In certain preferred embodiments,nucleotides comprise a detectable label that is indicative of the basecomposition of the nucleotide such that a signal emitted from the labelidentifies the base present in the nucleotide. As such, multiple typesof differentially labeled nucleotides can be present in a sequencingreaction, and detection and identification of signals emitted fromnucleotides incorporated into a nascent strand allows determination ofthe base sequence of the nascent strand and, by complementarity, thebase sequence of the template nucleic acid. In certain preferredembodiments, a label is attached to a nucleotide at a phosphate groupthat is removed from the nucleotide upon incorporation into the nascentstrand, e.g., at a terminal phosphate.

In certain aspects, polymerase reactions are monitored in real time,e.g., during processive incorporation of nucleotides (or analogsthereof) into a nascent strand. For example, in certain embodiments,polymerase reactions of the invention are employed in a single-molecule,real-time sequence-by-incorporation process, e.g., as described in U.S.Pat. Nos. 6,753,200, 7,033,764, 7,056,661, and 7,056,676 and Levene etal., Science 299:682-686, January 2003 Zero-Mode Waveguides forSingle-Molecule Analysis at High Concentration, the full disclosures ofeach of which are hereby incorporated herein by reference in theirentireties for all purposes, and in particular for their teachingsregarding polymerase reactions employed in sequence-by-incorporationprocesses. Briefly, a polymerase/template/primer complex is providedimmobilized upon a surface of a substrate, and the complex is exposed toa complete set of four nucleotides or nucleotide analogs. The analogsare labeled such that incorporation of each nucleotide into theextending primer releases the label from the nucleotide. Preferrednucleotide and nucleotide analogs are described in e.g., theabove-incorporated patents, and U.S. Pat. Nos. 7,041,812 and 7,052,839,the full disclosures of which are incorporated herein by reference intheir entireties for all purposes and in particular for their teachingsregarding such nucleotides and nucleotide analogs and their use insequencing-by-incorporation methods.

In an exemplary embodiment, the polymerase reaction is isolated withinan extremely small observation volume that effectively results inobservation of individual polymerase molecules. As a result, anincorporation event provides observation of an incorporating nucleotideanalog that is readily distinguishable from non-incorporated nucleotideanalogs. In a further exemplary embodiment, such small observationvolumes are provided by immobilizing the polymerase enzyme within anoptical confinement, such as a Zero Mode Waveguide (“ZMW”). For adescription of ZMWs and their application in single molecule analyses,and particularly nucleic acid sequencing, see, e.g., Published U.S.Patent Application No. 2003/0044781, and U.S. Pat. No. 6,917,726, eachof which is incorporated herein by reference in its entirety for allpurposes, and in particular for its teachings regarding ZMWs. Suchsequential reaction and monitoring permits a real-time monitoring of thepolymerization reaction and determination of the sequence of thetemplate nucleic acid.

FIG. 1 provides an illustrative example of a polymerase 105 immobilizedwithin a zero mode waveguide 110 during conversion of a single-strandedtemplate 115 into a double-stranded product 120. The small volume of thereaction refers to the volume of the zero mode waveguide in which thereaction is immobilized, but the volume of the entire reaction mixturefurther comprises the volume of the reaction mixture outside the zeromode waveguide.

In further exemplary embodiments, nucleic acid polymerase reactions ofthe invention include polymerase chain reactions (PCR), transcription,reverse transcription, nucleic acid sequence-based amplification(NASBA), self-sustained sequence replication (3SR), strand displacementamplification (SDA), and branched DNA signal amplification (hDNA)reactions.

Reduction of Steric Hindrance in Nucleic Acid Polymerase Reactions

As a nucleic acid polymerase reaction proceeds, the resultant growingnascent nucleic acid strand can interfere with the progression of thepolymerase enzyme—this interference is also referred to herein as“steric hindrance.” Such steric hindrance can be especially problematicin small volume reactions, because the growing nascent strand is morelikely to encounter and hinder the polymerase enzyme than would be thecase in reactions taking place in larger volumes. The present inventionprovides methods and compositions for reducing and/or preventing thesteric hindrance that can be caused by the product of a nucleic acidpolymerase reaction. In certain embodiments, steric hindrance is reducedor prevented by cleaving and/or removing at least one or more portionsof the growing nascent strand, e.g., by cleavage of the sugar-phosphatebackbone or by removal of a base to create an abasic site, whichenhances flexibility and is more susceptible to hydrolysis than anunaltered nucleotide. As such, in preferred embodiments the nascentstrand produced by a polymerase during a polymerase reaction is renderedunsuitable for use as a single template in a subsequent polymerasereaction, e.g., due to fragmentation and/or other modification.

Using Exonucleases

In certain aspects, the present invention provides methods andcompositions for reducing steric hindrance caused by the product of anucleic acid polymerase reaction by cleaving that product using anexonuclease. An exonuclease is any of various enzymes that cleavenucleotides sequentially from free ends of a nucleic acid substrate. Inone exemplary embodiment, a single-stranded nascent nucleic acidproduced in nucleic acid polymerase reaction is cleaved or “degraded”with an exonuclease, e.g., a ssDNA exonuclease. In another aspect, adouble-stranded molecule comprising the nascent nucleic acid strandannealed to its complementary region on the template nucleic acid iscleaved with an exonuclease, e.g., a dsDNA exonuclease possessing 5′→3′exonucleases activity, 3′→5′ exonucleases activity, or both.

In certain embodiments, the exonuclease cleaves the product of thenucleic acid polymerase reaction without cleaving the template nucleicacid. For example, the exonucleases may degrade a single-strandedproduct produced by strand-displacement of the nascent strand from acircular template. In other cases, the cleavage may occur while thenascent strand is still bound to the template strand, but may bespecific for the nascent strand, e.g., due to the polarity of theexonucleases activity. In other embodiments, the exonuclease cleaves thetemplate nucleic acid, but only in regions that have already been usedby the polymerase enzyme in the nucleic acid synthesis reaction.

Various exonucleases can be used in accordance with the presentinvention. Such exonucleases are well known in the art. Exemplaryexonucleases for use in the present invention include withoutlimitation: RecJ, lambda exonuclease, T7 exonuclease, exonuclease I,exonuclease III, exonuclease T, and the like. A single type ofexonuclease may be utilized according to the invention, or a combinationof two or more exonucleases may be used. Certain preferred exonucleasesdegrade only one strand of a double-strand DNA in the 5′ to 3′direction.

In accordance with the invention, the exonuclease may be usedsimultaneously with a nucleic acid polymerase in a polymerase reaction,such that new nucleotides are being added to the 3′ end of the nascentnucleic acid strand by the polymerase enzyme while, at the same time,nucleotides are being removed from the 5′ end of the same nascentnucleic acid strand by the exonuclease. In certain embodiments, theexonuclease initiates at only the 5′ end of the nascent nucleic acidstrand. In another embodiment initiates at only the 3′ end of thetemplate nucleic acid strand. In still another embodiment, theexonuclease is able to initiate at either the 3′ end of the templatestrand or 5′ end of the nascent nucleic acid strand. In still furtherembodiments, the exonuclease cleaves both strands of a double-strandedproduct comprising both the nascent strand and the template strand. Inone exemplary embodiment, an exonuclease is present in the reactionmixture at the start of a nucleic acid polymerase reaction of theinvention. In another exemplary embodiment, an exonuclease is introducedafter initiation of the nucleic acid polymerase reaction, e.g., afterthe reaction has proceeded for a period of time.

In certain preferred embodiments, exonucleases can be used duringsequencing reactions, such as sequencing-by-incorporation reactions, toreduce and/or prevent steric hindrance of polymerase enzyme activity bythe growing nascent strand. Such sequencing-by-incorporation reactionsare well known in the art and described further herein. In one exemplaryembodiment, a sequencing reaction in accordance with the presentinvention will include the step of conducting a nucleic acid polymerasereaction in the presence of at least one detectableterminal-phosphate-labeled nucleotide and at least one exonuclease. Insuch an embodiment, the sequencing reaction results in addition of thedetectable terminal-phosphate-labeled nucleotide to a nascent nucleicacid strand. In a further embodiment, the reaction also results inproduction of a labeled polyphosphate that is released from thedetectable terminal-phosphate-labeled nucleotide. In this embodiment,the released labeled polyphosphate or the terminal-phosphate-labelednucleotide are detected and can be identified, and this identificationcan allow determination of the nucleotide sequence of the templatenucleic acid. In certain embodiments, such as those in which a templateis repeatedly processed by the polymerase to produce one or more nascentstrands comprising one or more copies of the complement of the template,the exonuclease included in the reaction preferably cleaves the nascentnucleic acid strand but does not cleave the template nucleic acid.

FIG. 2 provides exemplary embodiments of certain single moleculesynthesis reactions comprising an exonuclease. The diagram in FIG. 2Aillustrates a polymerase enzyme 215 processing a linear, single-strandedtemplate 210, thereby synthesizing a complementary nascent strand 225. AdsDNA exonuclease 220 possessing exonuclease activity degrades thenascent strand from the 3′ terminus during synthesis of the 5′ terminusof the nascent strand by the polymerase 215. Although a 5′→3′ dsDNAexonuclease is shown, optionally a exonucleases could be used, e.g., inapplications in which preservation of the template nucleic acid is notnecessary or desired. FIG. 2B also depicts a polymerase enzyme 215processing a linear, single-stranded template 210 and synthesizing acomplementary nascent strand 225. However, the dsDNA exonuclease 230possesses both 5′→3′ and 3′→5′ exonuclease activity that degrades boththe nascent strand and the template strand during nascent strandsynthesis by the polymerase 215. FIG. 2C illustrates a polymerase enzyme260 that possesses strand displacement activity processing a circular,single stranded template 250 to synthesize a concatemericsingle-stranded nascent strand 255. A ssDNA exonucleases 240 degradesthe nascent strand from the 3′ terminus after it has been displaced bythe translocating polymerase 260. In each embodiment depicted in FIG. 2,steric hindrance in a polymerase reaction is alleviated by cleavage ofthe product of the polymerase reaction by an exonuclease.

Using Restriction Endonucleases

In certain aspects, the invention provides methods and compositions forreducing steric hindrance caused by the product of a nucleic acidpolymerase reaction by utilizing one or more restriction endonucleasesto cleave that product. In certain exemplary embodiments, the nascentnucleic acid strand produced in the nucleic acid polymerase reaction iscleaved with a restriction endonuclease. In other embodiments, thedouble-stranded nucleic acid molecule comprising the nascent nucleicacid strand annealed to the complementary region on the template nucleicacid upon which it was synthesized is cleaved with a restrictionendonuclease. In a further aspect, the restriction endonuclease cleavesthe product of the nucleic acid polymerase reaction, but only in regionsof the template nucleic acid that have already been used by thepolymerase enzyme to add a nucleotide to the growing nascent strand in atemplate-dependent manner.

A restriction endonuclease is any of a group of enzymes that catalyzesthe cleavage of DNA molecules in a sequence-specific manner. Variousrestriction endonucleases can be used in accordance with the presentinvention. Such restriction endonucleases are well known andcharacterized in the art. In one exemplary embodiment, restrictionendonucleases with four-base-pair recognition sites are used inaccordance with the invention. In another non-limiting exemplaryembodiment, a restriction endonuclease utilized in accordance with thepresent invention is selected from a group that includes: AatII, Acc65I,AceI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI,AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII,AvrII, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI,BciVI, BelI, BfaI, BfuAI, BglI, BglII, BlpI, Bme1580I, BmgBI, BmrI,BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI,BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI,BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI,BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSI, BstAPI, BstBI,BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsCI,BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-I, CviQI, DdeI, DpnI, DpnII,DraI, DraIII, DrdI, EaeI, EagI, EarI, EciI, EcoNI, EcoO109I, EcoP15I,EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspI, HaeII, HaeIII, HgaI,HhaI, HincII, HindIII, HinfI, HinPII, HpaI, HpaII, HphI, Hpy188I,Hpy188III, Hpy99I, HpyCH4III, HpyCH4IV, HpyCH4V, KasI, KpnI, MboI,MboII, MfeI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspAII, MspI,MwoI, NaeI, NarI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NolI,NruI, NsiI, NspI, PacI, PaeR7I, PciI, PflFI, PflMI, PhoI, PleI, PmeI,PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI,RsrII, SacI, SacI, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScrFI, SexAI,SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI,StuI, StyD4I, StyI, SwaI, Taq^(α)I, TfiI, TliI, TseI, Tsp45I, Tsp509I,TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, and ZraI. A singletype of restriction endonuclease may be utilized according to theinvention, or a combination of two or more such endonucleases may beused.

In accordance with the invention, restriction endonucleases can be usedduring sequencing reactions, such as sequencing-by-incorporationreactions, to reduce and/or prevent any steric hindrance of polymeraseenzyme activity by the growing nascent strand. Suchsequencing-by-incorporation reactions are well known in the art anddescribed further herein. In accordance with the invention, therestriction endonuclease may be used simultaneously with a nucleic acidpolymerase in a polymerase reaction, such that new nucleotides are beingadded to the 3′ end of the nascent nucleic acid strand by the polymeraseenzyme while, at the same time, cleavage of the same nascent nucleicacid strand is carried out by the restriction endonuclease at one ormore positions within a product of the reaction comprising the nascentnucleic acid strand, e.g., that has exited the polymerase complex. Incertain embodiments, the restriction endonuclease cleaves only thenascent nucleic acid strand. In other embodiments, the restrictionendonuclease cleaves only the template strand when duplexed with thenascent strand. In other embodiments, the restriction endonucleasecleaves both strands of a double-stranded product comprising both thenascent strand and the template strand. In one exemplary embodiment, arestriction endonuclease is present in the reaction mixture at the startof a nucleic acid polymerase reaction of the invention. In anotherexemplary embodiment, a restriction endonuclease is introduced afterinitiation of the nucleic acid polymerase reaction, e.g., after thereaction has proceeded for a period of time.

In one exemplary aspect, a sequencing reaction in accordance with thepresent invention will include the step of conducting a nucleic acidpolymerase reaction in the presence of at least one detectableterminal-phosphate-labeled nucleotide and at least one restrictionendonuclease. In one such embodiment, the sequencing reaction results inaddition of the detectable terminal-phosphate-labeled nucleotide to anascent nucleic acid strand. In a further embodiment, the reaction alsoresults in production of a labeled polyphosphate that is released fromthe detectable terminal-phosphate-labeled nucleotide. In such anembodiment, the released labeled polyphosphate or theterminal-phosphate-labeled nucleotide can be detected and subsequentlyidentified, and this identification can allow determination of thenucleotide sequence of the template nucleic acid. In certainembodiments, such as those in which a template is repeatedly processedby the polymerase to produce one or more nascent strands comprising oneor more copies of the complement of the template, a restrictionendonuclease included in the reaction preferably cleaves the product ofthe polymerase reaction but does not cleave the template nucleic acid.

FIG. 3 provides exemplary embodiments of certain single moleculesynthesis reactions comprising an endonuclease. The diagram in FIG. 3Aillustrates a polymerase enzyme 315 processing a linear, single-strandedtemplate 310, thereby synthesizing a complementary nascent strand 325. AdsDNA-dependent ssDNA endonuclease 320 cleaves the sugar-phosphatebackbone of the nascent strand 325 and the template strand 310 in asequence-specific manner during synthesis of the nascent strand by thepolymerase 315. Although both the template and nascent strand are shownto be susceptible to cleavage by the endonuclease, in some embodimentscleavage is restricted to the nascent strand, e.g., in applications inwhich preservation of the template nucleic acid is necessary and/ordesirable as further described below. FIG. 3B also depicts a polymeraseenzyme 315 processing a linear, single-stranded template 310 andsynthesizing a complementary nascent strand 325. However, the dsDNAendonuclease 330 cleaves the sugar-phosphate backbone of both thenascent strand and the template strand at or proximal to a recognitionsite during nascent strand synthesis by the polymerase 315. FIG. 3Cillustrates a polymerase enzyme 360 that possesses strand displacementactivity processing a circular, single stranded template 350 tosynthesize a concatemeric single-stranded nascent strand 355. A ssDNAendonuclease 340 degrades the nascent strand in a sequence-specificmanner after it has been displaced by the translocating polymerase 360.Although this rolling-circle synthesis reaction is shown with a ssDNAendonuclease 340, it could also or alternatively include adsDNA-dependent ssDNA endonuclease. Such an embodiment would preferablyinclude modification of the template strand to protect it fromendonucleolytic cleavage, thereby promoting greater processivity of thepolymerase enzyme by preservation of the template on which it istranslocating. Methods for protecting a template strand from cleavageare further discussed below. In each embodiment depicted in FIG. 3,steric hindrance in a polymerase reaction is alleviated by cleavage ofthe product of the polymerase reaction by an endonuclease.

Using Uracil-DNA Glycosylases

In one aspect, the present invention provides methods for reducingsteric hindrance caused by the product of a nucleic acid polymerasereaction by modifying that product using a uracil-DNA glycosylase. Auracil-DNA glycosylase is any of various enzymes that remove uracilnitrogenous bases from single-stranded or double-stranded DNA withoutdestroying the sugar-phosphate backbone. The resulting abasic sites(also termed “apurinic/apyrimidinic (AP) sites”) are susceptible tohydrolytic cleavage, so removal of uracil bases is usually accompaniedby fragmentation of the DNA. Further, the introduction of abasic sitesadds flexibility to the nascent strand, thereby mitigating sterichindrance to some extent even absent fragmentation. Since uracil-DNAglycosylases can act on single-stranded or double-stranded DNA, uracilresidues can be removed from the nascent strand both when bound to thetemplate strand or when rendered single-stranded, e.g., by stranddisplacement of the nascent strand from the template strand.

In certain embodiments, a nucleic acid that does not comprise dUTPnucleotides is a template in a single-molecule sequencing reaction inthe presence of dUTP nucleotides. The dUTP nucleotides can entirelyreplace dTTP residues in the reaction mixture, or a combination of dUTPand dTTP may be present, as described in more detail below. As apolymerase processes the template, nucleotides comprising uracilnucleobases are incorporated into the nascent strand, but the nucleotidecomposition of the template strand remains unchanged. This underscoresone additional advantage of the methods comprising incorporation andsubsequent removal of uracil residues, which is that the template strandneed not be modified to be preserved during the course of the reaction.As such, the template can be used multiple times in the same oradditional polymerase reactions without any “protective” chemicalmodifications, which are desired in certain embodiments of the inventioncomprising nuclease degradation of the nucleic acid product. Forexample, certain embodiments comprise repeated processing of a templatenucleic acid by the polymerase to produce one or more nascent strandscomprising one or more copies of the complement of the template. By onlymodifying the nascent strand, the template strand is preserved foradditional nascent strand generation by the same or a differentpolymerase enzyme.

DNA-uracil glycosylases remove uracil residues from single- anddouble-stranded DNA without cleavage of the sugar-phosphate backbone.Various uracil-DNA glycosylases can be used in accordance with thepresent invention. Such uracil-DNA glycosylases are well known in theart. Exemplary uracil-DNA glycosylases for use in the present inventioninclude without limitation: E. coli UDG, marine bacterium BMTU 3346 UNG,Archaeoglobus fulgidus UDG and H. sapiens UNG, all of which arecommercially available, e.g., from Roche Applied Science, New EnglandBiolabs, Invitrogen, Affymetrix, and/or Fermentas Life Sciences. Asingle type of uracil-DNA glycosylase may be utilized according to theinvention, or a combination of two or more uracil-DNA glycosylases maybe used.

In accordance with the invention, the uracil-DNA glycosylase may be usedsimultaneously with a nucleic acid polymerase in a polymerase reaction,such that new nucleotides are being added to the 3′ end of the nascentnucleic acid strand by the polymerase enzyme while, at the same time,uracils are being removed from the same nascent nucleic acid strand bythe uracil-DNA glycosylase. In certain embodiments in which a DNAmolecule is the template nucleic acid, the uracil-DNA glycosylase actsonly on the nascent nucleic acid strand since a DNA template istypically lacking uracil residues. In one exemplary embodiment, auracil-DNA glycosylase is present in the reaction mixture at the startof a nucleic acid polymerase reaction of the invention. In anotherexemplary embodiment, a uracil-DNA glycosylase is introduced afterinitiation of the nucleic acid polymerase reaction, e.g., after thereaction has proceeded for a period of time.

In certain preferred embodiments, uracil-DNA glycosylases can be usedduring sequencing reactions, such as sequencing-by-incorporationreactions, to reduce and/or prevent steric hindrance of polymeraseenzyme activity by the growing nascent strand. Suchsequencing-by-incorporation reactions are well known in the art anddescribed further herein. In one exemplary embodiment, a sequencingreaction in accordance with the present invention will include the stepof conducting a nucleic acid polymerase reaction in the presence of atleast one detectable terminal-phosphate-labeled nucleotide, dUTP (or ananalog or derivative thereof), and at least one uracil-DNA glycosylase.In such an embodiment, the sequencing reaction results in addition ofthe detectable terminal-phosphate-labeled nucleotide and dUTP into anascent nucleic acid strand. In a further embodiment, the reaction alsoresults in production of a labeled polyphosphate that is released fromthe detectable terminal-phosphate-labeled nucleotide. In thisembodiment, the released labeled polyphosphate or theterminal-phosphate-labeled nucleotide are detected and can beidentified, and this identification can allow determination of thenucleotide sequence of the template nucleic acid. The uracil-DNAglycosylase will act on the double-stranded product of the sequencingreaction that comprises the nascent strand and the template on which itwas assembled. Uracil residues in this product are removed by theuracil-DNA glycosylase producing abasic sites. The presence of abasicsites in the double-stranded or single-stranded product increasesflexibility, which can ease steric hindrance caused by the elongatingproduct in the restricted volume reaction vessel.

The resulting abasic sites are susceptible to hydrolytic cleavage, whichcauses fragmentation of the strand comprising the abasic site, e.g., inthe nascent strand. The hydrolytic cleavage of the abasic strand can beincreased by altering reaction conditions, e.g., by increasing thereaction temperature or by addition of reaction components that promotehydrolysis. For example, an AP endonuclease can be included in thereaction mixture to ensure fragmentation of the strand containing the APsites. For example, Class II AP endonucleases catalyze the hydrolysis ofthe 5′-phosphodiester of an abasic site to generate a 3′-OH group and a5′-abasic residue. (For more information on AP endonucleases, sec, e.g.,Wilson, et al. (1997) Nuc. Ac. Res. 25(5):933-939, and Demple, et al.(1994) Annu. Rev. Biochem. 63:915-948, both of which are incorporated byreference herein in their entireties for all purposes.) Further,hydrolysis of apurinic sites in DNA can be induced by polyamines andother amines, such as spermine, spermidine, putrescine, and cadaverine.Further information on polyamine-induced hydrolysis of abasic sites canbe found in the art, e.g., Male, et al. (1982) Nuc. Ac. Res.10(20):6305-6318; Lindahl, et al. (1972) Biochemistry 11:3618-3623;Tamm, et al. (1952) J. Biol. Chem. 195:40-63; and Tamm, et al. (1952) J.Biol. Chem. 199:313-327, the disclosures of all of which areincorporated herein by reference in their entireties for all purposes.

FIG. 4 provides exemplary embodiments of certain single moleculesynthesis reactions comprising a uracil-DNA glycosylase. The diagram inFIG. 4A illustrates a polymerase enzyme 415 processing a linear,single-stranded template 410, thereby synthesizing a complementarynascent strand 425. A uracil-DNA glycosylase 420 cleaves theN-glycosylic bond in dUTP to remove the uracil base and generate anabasic (AP) nucleotide (405) in the nascent strand 425 during synthesisof the nascent strand by the polymerase 415. In preferred embodiments,only the nascent strand comprises uracil residues that were incorporatedby the polymerase during single-molecule synthesis reaction, andmodification by the uracil-DNA glycosylase is therefore restricted tothe nascent strand. FIG. 4B illustrates a polymerase enzyme 460 thatpossesses strand displacement activity processing a circular, singlestranded template 450 to synthesize a concatemeric single-strandednascent strand 455. A uracil-DNA glycosylase 430 removes uracil residuesfrom the nascent strand 455 after it has been displaced by thetranslocating polymerase 460. The abasic sites in the nascent strand aresusceptible to hydrolysis, after which the nascent strand is fragmented.Methods for promoting hydrolysis of abasic sites are well known in theart and further described elsewhere herein. Although this rolling-circlesynthesis reaction is shown with the uracil-DNA glycosylase acting onlyon the single-stranded portion of the nascent strand, uracil-DNAglycosylases can also act on double-stranded DNA so uracil residues canalso be removed from the nascent strand when bound to the templatestrand. In each embodiment depicted in FIG. 4, steric hindrance in apolymerase reaction is alleviated by modification of the product of thepolymerase reaction by a uracil-DNA glycosylase, e.g. by the increasedflexibility of a nucleic acid comprising abasic sites and by the highersusceptibility of the nascent strand to fragmentation.

Protection of Template Nucleic Acid

In accordance with the invention, it can be advantageous to protect thetemplate nucleic acid from methods and compositions used to alleviateand/or prevent steric hindrance from the product of a polymerasereaction. Such protection allows the polymerase reaction to continuewith the intact template nucleic acid while the product of thepolymerase reaction is cleaved and/or removed. For example, a polymerasecan continue around a circular template multiple times to generate along, concatemeric nascent strand comprising multiple copies of thecomplement of the template strand. Further, an immobilized lineartemplate can be repeatedly processed by the same polymerase, or can beprocessed by multiple different polymerases, in some embodimentssimultaneously. Damage to the template that prevents further processingby a polymerase would prevent such repetitive processing of the template(absent any means to repair the template).

In certain aspects, the template nucleic acid used in nucleic acidpolymerase reactions according to the invention is modified such that arestriction endonuclease or exonuclease is unable to cleave the modifiedtemplate nucleic acid. For example, in certain embodiments templatenucleic acids of the invention are modified to comprise at least onenon-natural nucleotide at one or both termini. Such non-naturalnucleotides are well known in the art and include without limitation:peptide nucleic acids (PNAs), locked nucleic acids (LNA),oligonucleotide N3′→P5′ phosphoramidates,oligo-2′-O-alkylribonucleotides, and the like. In other exemplaryembodiments, template nucleic acids of the invention are methylated.Such a modification can prevent a restriction endonuclease from cleavingthe template nucleic acid.

In still other exemplary embodiments, template nucleic acids of theinvention are modified to include a protecting group on at least onenucleotide. Such a protecting group may in some embodiments be aremovable protecting group. In further embodiments, such protectinggroups comprise a chemically modified oligonucleotide extension ligatedto an end of the template nucleic acid. As used herein, a chemicallymodified oligonucleotide extension comprises a short polymer ofnucleotides, wherein one or more of the nucleotides are altered in waysknown in the art. For example, such chemically modified oligonucleotideextensions can include phosphorothioate bonds or spacers such as C3, 9,C12 and 18 (Glen Research). 3′-Spacer C3 CPG may act as a blocker ofexonuclease activity at the 3′-terminus. In one embodiment, Spacer 18has a structure according to the formula:

In other exemplary embodiments, template nucleic acids can be protectedusing a 5′-Aldehyde-Modifier C2 Phosphoramidite (Glen Research) or a9-fluorenylmethoxycarbonyl (Fmoc) protecting group.

In certain exemplary embodiments, the template nucleic acids areprotected with a removable protecting group that is a photoremovableblocking group. Such photoremovable blocking groups can in one exemplaryembodiment of the invention be incorporated with a triphosphatenucleotide as the last base unit on the template nucleic acid. Suchincorporation can be achieved in one non-limiting example byenzymatically using a DNA polymerase to give the targeted nucleic acid aphotoremovable blocking group.

A number of different types of photoremovable chemical blocking groupshave been described in the art. In general, such groups include, e.g.,nitroveratryl, 1-pyrenylmethyl, 6-nitroveratryloxycarbonyl,dimethyldimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,methyl-6-nitropiperonyloxycarbonyl, 2-oxymethylene anthraquinone,dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl,o-hydroxy-alpha-methyl cinnamoyl, and mixtures thereof, the compositionsand applications of which are described in, e.g., U.S. Pat. Nos.5,412,087, 5,143,854, 6,881,836, Albert et al., Nucl. Acids Res, (2003)31(7):e35, Beier et al., Nucleic Acids Res. (2000) 28(4):el I, Pon etal, Nucleic Acids Res. (2004) 32(2):623-631, Olejnik et al., NucleicAcids Res. (1998) 26(15):3572-3576, and Blanc et al. J. Org. Chem.(2002) 67:5567-5577, each of which is incorporated herein by referencein its entirety for all purposes, and in particular for teachingsrelated to photoremovable chemical blocking groups.

Other methods and compositions for protecting template nucleic acids arewell known in the art and can be used in accordance with the presentinvention.

Compositions of the Invention

In certain aspects, the invention provides a composition that includes anucleic acid polymerase, a substrate, one or more restrictionendonucleases or exonucleases, and a template nucleic acid. In certainexemplary embodiments, one or more of the nucleic acid polymerase, thetemplate nucleic acid and the restriction endonuclease or exonucleaseare immobilized on the surface of the substrate. In some embodiments,the substrate comprises one or more zero mode waveguides or otheroptical confinements. In further embodiments, the template nucleic acidis modified as described further herein such that the restrictionendonuclease or exonuclease is unable to cleave the template nucleicacid. In still further embodiments, the composition further includes atleast four different nucleotides or nucleotide analogs, and in preferredembodiments, the at least four nucleotides or nucleotide analogs arelabeled. For example, one or more of the at least four nucleotides ornucleotide analogs are preferably differentially labeled such thatsignals detected from each are distinct from one another, and the signalis indicative of the type of nucleotide bearing the label.

In certain aspects, the invention provides a composition that includes anucleic acid polymerase, a substrate, one or more uracil-DNAglycosylases, and a template nucleic acid. In certain exemplaryembodiments, one or more of the nucleic acid polymerase, the templatenucleic acid and the one or more uracil-DNA glycosylases are immobilizedon the surface of the substrate. In some embodiments, the substratecomprises one or more zero mode waveguides or other opticalconfinements. In certain embodiments, the composition further includesat least four different nucleotides or nucleotide analogs, wherein atleast one is dUTP or a derivative or analog thereof. In preferredembodiments, the at least four nucleotides or nucleotide analogs arelabeled. For example, one or more of the at least four nucleotides ornucleotide analogs are preferably differentially labeled such thatsignals detected from each are distinct from one another, and the signalis indicative of the type of nucleotide bearing the label. In certainembodiments, the composition comprises at least five differentnucleotides or nucleotide analogs, wherein at least one is dUTP (whichincludes derivatives or analogs thereof) and at least one other is dTTP(which includes derivatives or analogs thereof). For example, acomposition of the invention can include a ratio of dUTP:dTTP equal toabout 20:1, 15:1, 10:1, 7.5:1, 5:1, 4:1, 3:1, 2:1, 1:1, 2:1, 3:1, 4:1,5:1, 7.5:1, 10:1, 15:1, or 20:1. In other embodiments, the concentrationof dUTP may be between 20- and 100-fold higher than the concentration ofdTTP, and vice versa. In certain embodiments, a ratio of dUTP:dTTP isselected to ensure sufficient modification of the reaction product toalleviate steric hindrance while allowing efficient nucleotideincorporation into the nascent strand and processive polymeraseactivity. In certain embodiments, a composition of the inventioncomprises at least one uracil-DNA glycosylase and at least onepolyamine. In other embodiments, a composition of the inventioncomprises at least one uracil-DNA glycosylase and at least one APendonuclease. In yet further embodiments, a composition of the inventioncomprises at least one uracil-DNA glycosylase and a buffer that enhanceshydrolysis at abasic sites in the nascent strand.

As will be appreciated, substrates that are part of compositions of theinvention can comprise any material capable of supporting one or moremolecules of the invention, including nucleic acid polymerases,restriction endonucleases, exonucleases, and template nucleic acids.Exemplary substrate materials include, but are not limited to, inorganiccrystals, inorganic glasses, inorganic oxides, metals, organic polymersand combinations thereof. Inorganic glasses and crystals of use in thesubstrate include, but are not limited to, LiF, NaF, NaCl, KBr, Kl,CaF₂, HgF₂, BN, AsS₃, ZnS, Si₃N₄ and the like. The crystals and glassescan be prepared by art standard techniques. See, for example, Goodman,CRYSTAL GROWTH THEORY AND TECHNIQUES, Plenum Press, New York 1974.Alternatively, the crystals can be purchased commercially (e.g., FischerScientific). Inorganic oxides of use in the present invention include,but are not limited to, Cs₂O, Mg(OH)₂, TiO₂, ZrO₂, CeO₂, Y₂O₃, Cr₂O₃,Fe₂O₃, NiO, ZnO, Al₂O₃, SiO₂ (glass), quartz, In₂O₃, SnO₂, PbO₂ and thelike. Metals of use in the substrates of the invention include, but arenot limited to, gold, silver, platinum, palladium, nickel, copper andalloys and composites of these, metals.

Kits of the Invention

In certain aspects, the present invention provides kits for theprevention of steric hindrance during nucleic acid polymerase reactions.In certain exemplary embodiments, such kits include a substrate, whichcomprises a surface, a nucleic acid polymerase, and a restrictionendonuclease and/or an exonuclease. In further exemplary embodiments,one or more of the restriction endonuclease and/or exonuclease and thenucleic acid polymerase are immobilized on the surface of the substrate.In certain exemplary embodiments, such kits include a substrate, whichcomprises a surface, a nucleic acid polymerase, and a uracil-DNAglycosylase. In further exemplary embodiments, one or more of theuracil-DNA glycosylase and the nucleic acid polymerase are immobilizedon the surface of the substrate. In some embodiments, the kit includesat least four or five nucleotides or nucleotide analogs. In certainpreferred embodiments, one or more of the at least four or fivenucleotides or nucleotide analogs included in such a kit are labeled.For example, the at least four or five nucleotides or nucleotide analogsare preferably differentially labeled such that signals detected fromeach are distinct from one another, and the signal is indicative of thetype of nucleotide bearing the label.

Kits of the invention may further include any buffers, reagents andco-factors for use in nucleic acid polymerase reactions and/or withexonucleases, restriction endonucleases, and/or uracil-DNA glycosylasesused in accordance with the invention. Such buffers, reagents andco-factors are well-known in the art. It will be appreciated that thecombination of buffers, reagents and co-factors used in kits of theinvention will depend on the nucleic acid polymerases, restrictionendonucleases and exonucleases, uracil-DNA glycosylases used in thisaspect of the invention.

Kits of the invention may also include one or more reagents forprotecting a template nucleic acid from restriction endonuclease and/orexonuclease activity, according to the invention as described furtherherein. Such reagents are well-known in the art. Kits of the inventionmay also include one or more reagents for promoting hydrolysis at APsites in the nascent strand, according to the invention as describedfurther herein.

In certain aspects, the present invention provides methods foralleviating steric hindrance of polymerase reactions, and in particularpolymerase reactions confined to a restricted volume reaction vessel. Incertain aspects, compositions and kits are provided, e.g., for use incarrying out certain methods of the invention.

It is to be understood that although described in some detail forpurposes of illustration it is not intended to be restrictive. It willbe readily appreciated to one skilled in the art that a number ofvariations and modifications known or appreciated by those of skill inthe art may be practiced within the scope and spirit of presentinvention. The scope of the invention should, therefore, be determinednot with reference to the above description, but should instead bedetermined with reference to the appended claims, along with the fullscope of equivalents to which such claims are entitled. Unless otherwiseclear from the context or expressly stated, any concentration valuesprovided herein are generally given in terms of admixture values orpercentages without regard to any conversion that occurs upon orfollowing addition of the particular component of the mixture. Allpublications mentioned herein are cited for the purpose of describingand disclosing reagents, methodologies and concepts that may be used inconnection with the present invention. Nothing herein is to be construedas an admission that these references are prior art in relation to theinventions described herein. Throughout the disclosure various patents,patent applications and publications are referenced. To the extent notalready expressly incorporated herein, all published references andpatent documents referred to in this disclosure are incorporated hereinby reference in their entireties for all purposes.

We claim:
 1. A nucleic acid sequencing reaction mixture comprising: a. anucleic acid polymerase, wherein said nucleic acid polymerase isimmobilized on a substrate within a constricted volume reaction vessel;b. an enzyme selected from the group consisting of an endonuclease, anexonuclease, and a uracil-DNA glycosylase; and c. a template nucleicacid, wherein said enzyme is unable to modify said template nucleicacid.
 2. The composition of claim 1, further comprising a set ofdifferentially labeled nucleotides.
 3. The composition of claim 2,wherein the differentially labeled nucleotides comprise a label attachedat a phosphate group.
 4. The composition of claim 1, wherein theconstricted volume reaction vessel has a volume of less than oneattoliter.
 5. The composition of claim 1, wherein the constricted volumereaction vessel is a zero mode waveguide.
 6. The composition of claim 1,further comprising uracil-containing nucleotides.
 7. The composition ofclaim 6, further comprising thymine-containing nucleotides.
 8. Thecomposition of claim 6, further comprising at least one polyamine or APendonuclease.
 9. The composition of claim 1, wherein the templatenucleic acid comprises a modification that prevents cleavage by theexonuclease or the endonuclease.
 10. The composition of claim 1, whereinthe template nucleic acid is a circular nucleic acid template.
 11. Thecomposition of claim 1, wherein the template nucleic acid is asingle-stranded nucleic acid template.
 12. The composition of claim 1,wherein the template nucleic acid is a double-stranded nucleic acidtemplate.
 13. A kit comprising a. a substrate, wherein said substratecomprises at least one constricted volume reaction vessel comprising asurface; b. a nucleic acid polymerase, wherein the nucleic acidpolymerase can be immobilized on said surface of said constricted volumereaction vessel; c. a restriction endonuclease, an exonuclease, or auracil-DNA glycosylase.
 14. The kit of claim 13, further comprising aset of differentially labeled nucleotides.
 15. The kit of claim 13comprising the uracil-DNA glycosylase, further comprisinguracil-containing nucleotides.
 16. The kit of claim 13 comprising theexonuclease, wherein the exonuclease is a 5′→3′ ssDNA exonuclease. 17.The kit of claim 13 comprising the endonuclease, wherein theendonuclease is a restriction endonuclease.
 18. A method of increasingflexibility of a nascent strand, the method comprising (a) performing anucleic acid synthesis reaction to generate a nascent strand, and (b)creating abasic sites within the nascent strand, thereby increasing theflexibility of the nascent strand.
 19. The method of claim 18, whereinthe creating is performed using a uracil-DNA glycosylase.
 20. The methodof claim 18, further comprising cleaving the nascent strand at theabasic sites.